1. Technical Field of the Invention
The present invention involves the creation of defined chromosomal deficiencies, inversions and duplications using Cre recombinase in embryonic stem cells and transmitted into the mouse germ line. In the present invention, these chromosomal reconstructions can extend up to 3-4 cM. Chromosomal rearrangements are the major cause of inherited human disease and fetal loss. Further, chromosomal translocations and deletions are recognized as major genetic changes that are causally involved in neoplasia. Chromosomal variants such as deletions and inversions are exploited commonly as genetic tools in diploid organisms such as Drosophila. In diploid organisms, such deficiencies are exploited in genetic screens because a small portion of the genome is functionally hemizygous. Thus, a mutation which would normally be recessive and masked by the wildtype allele in a diploid context will be dominant and detectable in the haploid state. In the mouse, deficiencies have not, up to now, been available generally; thus, screens for recessive mutations are nonexistent or particularly cumbersome. However, the present invention provides methods to engineer mice and cell lines with defined regions of segmental haploidy. Such mice are useful for genetic screening and provide accurate models of human chromosomal diseases.
2. The Prior Art
Inherited chromosomal rearrangements such as inversions, duplications and deficiencies are responsible for a significant fraction of human congenital disease. Chromosomal changes also occur somatically and are associated with neoplastic disease. Defining the causal genetic alteration in a region of the genome associated with chromosomal rearrangements can be relatively straightforward if the affected gene lies in the breakpoint of an inversion or translocation. However, in cases of duplications and deficiencies, the specific genetic lesion(s) associated with pathological chromosomal changes are much harder to identify. Still, the generation of animal models that accurately recapitulate the genetic lesion would facilitate the study of disease and could be very helpful in the efforts to dissect specific gene-function relationships in multigene syndromes.
In diploid organisms such as Drosophila, chromosomal deficiencies are commonly exploited in genetic screens because a small portion of the genome is functionally hemizygous. Thus, a mutation which would be recessive and masked by the wildtype allele in the diploid context will be dominant and therefore readily detectable in the haploid state. In the mouse, deficiencies are not available generally. Despite the limited number of deficiencies available in the mouse, the potential for the detailed analysis of a genetic interval using these deficiencies has been demonstrated clearly. See Holdener-Kenny, et al., BioEssays, 14:831-39 (1992), which is hereby incorporated by reference.
Deficiencies that are available currently in the mouse genome were generated at random using ionizing irradiation. Although conventional gene targeting technology in embryonic stem (ES) cells can generate virtually any type of mutation, including deletions of up to 20 kb, it has not been possible to delete substantially larger fragments by using standard methodology. Likewise, the technology required to construct large inversions and duplications has not been established.
One mechanism by which chromosomes may be engineered is by the use of Cre recombinase. Cre recombinase has been used in mammalian cell lines and in vivo to delete or invert sequences between the 34 base pair recognition sequences, loxP sites, placed a few kb apart on the same chromosome. The recombination is initiated by Cre proteins which bind to 13-bp inverted regions in the loxP sites and promote synapses or joining of a pair of sites. Next, the Cre proteins catalyze strand exchange between the pair of sites within an asymmetric 8-bp central spacer sequence by concerted cleavage and rejoining reactions, involving a transient DNA-protein covalent linkage. Smith, et al., Nature Genetics, 9:376-385 (1995); Gu, et al., Science, 265:103-06 (1994) and Sauer, Nucl. Acids Res., 17:147-61 (1989) (both of these references are hereby incorporated by reference). Additionally, recombinases have been used to induce mitotic recombination between homologous and non-homologous chromosomes in Drosophila, plants and mammalian cells. Embryonic stem cell technology has become a powerful tool for defining the function of mammalian genes, but mainly has been restricted to the mutation of single genes. Replacement vectors have been used to construct deletions of up to 19 kb; however, utilizing the same strategy to construct larger deletions (&gt;60 kb) has not been successful. In the present invention, the generation and direct selection of deletions, duplications and inversions, ranging from 90 kb to 3-4 cM, in ES cells is demonstrated.